Anti-microbial compositions and methods with 3-(5-nitro-2-furyl)-pyrazoles

ABSTRACT

COMPOUNDS OF THE CLASS OF 5-AMINO-4-CARBAMOYL-3-(5NITRO-2:FURYL)-PYRAZOLE SUBSTITUTED IN 1-POSITION OF THE PYRAZOLE RING BY ALKYL OR HYDROXYALKYL HAVE ANTI-MICROBIAL PROPERTIES; THESE COMPOUNDS ARE ACTIVE INGREDIENTS OF PHARMACEUTICAL AND FEEDSTUFF COMPOSITIONS; THEY ARE USEFUL FOR THE TREATMENT OF MICROBIAL INFECTIONS AND FOR PROTECTING ORGANIC MATERIAL AGAINST MICROBIAL ATTACK; A TYPICAL EMBODIEMENT IS 5-AMINO-4-CARBAMOYL-1-METHYL-3-(5-NITRO2-FURYL)-YPYRAZOLE.

United States Patent Office 3,830,926 Patented Aug. 20, 1974 U.S. Cl.424-273 3 Claims ABSTRACT OF THE DISCLOSURE Compounds of the class of5-amino-4-carbamoyl-3-(5- nitro-2-furyl)-pyrazole substituted inl-position of the pyrazole ring by alkyl or hydroxyalkyl have anti-microbial properties; these compounds are active ingredients ofpharmaceutical and feedstuff compositions; they are useful for thetreatment of microbial infections and for protecting organic materialagainst microbial attack; a typical embodiment is5-amino-4-carbamoyl-1-methyl-3-(5-nitro- 2-furyl)-pyrazole.

CROSS-REFERENCE TO RELATED APPLICATION This is a division of Ser. No.43,585, filed June 4, 1970, now US. Pat. No. 3,682,956.

DETAILED DESCRIPTION The present invention relates to substituted5-amino-4- carbamoyl-3-(5-nitro-2-furyl)-pyrazoles with anti-microbialactivity. It further relates to pharmaceutical and feedstuffcompositions as well as to methods for the treatment of microbialinfections of mammals and to methods of protecting organic materialagainst microbial attack.

More particularly, the present invention pertains to compounds offormula wherein R is alkyl having at most five carbon atoms orhydroxyalkyl having from two to five carbon atoms.

If R is alkyl, then it may be a straightor branched chain alkyl group.Preferably it will contain from one to three carbon atoms. Examples ofalkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tertiarylbutyl and n-pentyl groups. If R is hydroxyalkyl, thenpreferably it contains two or three carbon atoms.

Nitrofuryl-pyrazoles of formula I may form salts with organic andinorganic acids. Examples of suitable acids include hydrochloric,hydrobromic, sulphuric, phosphoric, methanesulphonic, ethanedisulphonic,acetic, trichloroacetic, oxalic, succinic, maleic, fumaric, malic,tartaric, citric and mandelic acids.

Although the compounds produced by the present invention have beenascribed by formula I above, they may also be represented by thefollowing tautomeric formula and any specified compound of the presentinvention may occur in either these tautomeric forms or as a mixture ofboth of them. In this specification, however, the compounds of thepresent invention are regarded for purposes of clarity as having theformula I and are thus described and exemplified as beingS-nitrofuryl-pyrazole derivatives.

Compounds of the present invention are produced according to a firstprocess comprising hydrolysing a nitrofuryl-pyrazole derivative havingformula II,

wherein R is as previously defined.

The process may be carried out under conventional hydrolytic conditions,preferably those conventionally used in the acidic hydrolysis ofnitriles, for instance by treating with aqueous sulphuric acid within atemperature range of between 0 and 100 C. Preferably the hydrolysis iseffected at a reaction temperature of between and C. Preferably, thecompound of formula II is heated with a mixture of concentratedsulphuric acid and ethanol during a reaction time of from one to twohours, and at a temperature of from 90 to 100 C. The resulting reactionproduct may then advantageously be poured into an excess of water toprecipitate the desired S-nitrofurylpyrazole of formula I.

The starting compounds of formula II may be prepared by reacting thecorresponding S-nitrofuryl-nitrilimine, which in one of its mesomericforms may be represented by the formula III,

0 N i l c o u we R (111) with malononitrile.

The nitro-furyl-nitrilimine of formula III may conveniently begenerated, during the course of the reaction with malononitrile, bytreating the corresponding nitrofuryl-ahalo-hydrazone having the formulaIV,

NHR

wherein X is halogen, and R is as defined before with a base.

The process may, if desired, be effected in the presence of a furtherconventional hydrogen halide acceptor. The halogen present in thehalo-hydrazone of formula IV is preferably chlorine or bromine.

The nitrofuryl-u-halo-hydrazones of formula IV are new compounds. Theymay be produced, for example, by reacting the correspondingnitrofuryl-hydrazones having the formula V,

NHR v with an N-halo compound bearing the requisite halogen. Examples ofN-halo compounds which may be used, are N-halo benzotriazoles and N-halosuccinimides. Of the N-halo succinimides, the N-chloro compound may beused, but the N-bromo derivative is preferred.

According to a second process a compound falling under formula I, andhaving formula I,

wherein R is alkyl containing at most five carbon atoms,

is prepared by hydrolysing and deacylating a compound of formula VI,

I i, (VI) wherein R is alkyl, and R is as defined before by treating itwith a protonating agent in a polar solvent, and then hydrolising theprotonated intermediate.

OzN WFOONH:

wherein R" is hydroxyalkyl having from two to five carbon atoms, areprepared by hydrolysing and deacylating a compound having formula VII,

ON ON fll I N NHR:

.LOR: (VII) wherein R is lower acyl, and

A is alkylene having from two to live carbon atoms, by treating it witha protonating agent in a polar solvent and then hydrolysing theprotonated intermediate.

In the hydrolising and deacylating processes, the preferred protonatingreagent is ethanolic hydrogen chloride, 40% w./w. being a suitableconcentration. Other protonating agents, such as sulphuric acid in otherpolar solvents, may also be used.

The second and third processes according to the invention areessentially similar. The only dilference is that in the second processthe substituent R as alkyl group is unchanged, whereas in the thirdprocess the substituent AOR is itself deacylated to form a hydroxy alkylgroup.

Normally both the second and third processes according to the inventionwill involve heating the starting material with a protonating agent suchas ethanolic hydrogen chloride for a period of time sufiicient to ensurereaction, then cooling the reaction mixture and either adding ice/water, or pouring the reaction mixture into ice/water to precipitate thedesired product. The processes are carried out at temperatures between 0C. and the boiling point of the polar solvent.

The starting materials of formula VI used for the second processaccording to the invention, may be prepared by acylating a compound offormula II,

0 l C, 2 W i N\N/NH2 wherein R is alkyl having at most five carbon atomswith an acylating agent containing the acyl group R The startingmaterials of formula VII used in the third process according to theinvention, are novel compounds and are found themselves to possessanti-microbial activity. These compounds of formula VII may be preparedby acylating with a suitable acylating agent compounds of formula II",

wherein R" is hydroxy alkyl having from two to five carbon atoms, inorder to introduce the acyl group R These compounds of formula II" maybe prepared by a similar series of reactions as described above,starting from a suitable substituted nitrofuryl nitrilimine.

The compounds of the present invention of formula I and formula VII havevaluable anti-microbial properties, in particular have anti-bacterial,anthelminthic, antiprotozoal cocidiostatic, trypanocidal andanti-malarial activity of value in human or veterinary medicine. Thecompounds are particularly valuable in the treatment of infections ofthe intestinal and urinary tracts. The compounds may also be used toprotect a high molecular weight hydrophobic or other organic materialsusceptible to bacterial or other microbial deterioration by contactingthe organic material with, impregnating in, or otherwise treating with,the compounds. The compounds also find application as growth-promotingadditives to animal feedstutfs.

The anti-microbial properties of the compounds of the invention aredemonstrated in a variety of standard in vitro and in vivo tests. Thus,5-amino-4-carbamoyl-lmethyl-3- S-nitro-Z-furyl -pyrazole and 5-amino-4-carbamoyl- 1- (2 hydroxyethyl) -3- 5-nitro-2-furyl -pyrazolehave been found to have an excellent activity against Staphylococcus,Escherichia coli, Klebsiella, Salmonella and other bacteria.

The toxicity of the compounds of the invention as determined in mice onoral administration is of favourably low order.

For their internal use in mammals, the compounds of formula I areadministered orally in daily dosages of from about 1 to about 50mg./kg., although the exact dosage has to be adjusted to the type ofinfection, the age, weight and the particular condition of the hostbeing treated.

The compounds of the present invention are administered advantageouslyin form of a pharmaceutical composition comprising an anti-microbiallyeffective amount of a compound of formula I and a pharmaceuticallyacceptable carrier therefor.

The pharmaceutical compositions according to the invention contain atleast one compound of formula I as active substance together with aconventional pharmaceutical carrier. The type of carrier actually useddepends to a great extent on the intended application. For externalapplication, for example in disinfecting healthy skin, disinfectingwounds and in treating dermatoses and infections of the mucous membranescaused by bacteria, ointments, powders and tinctures are particularlyuseful. The ointment bases may be anhydrous, for instance they canconsist of mixtures of wool fat and soft paraffin, or they can consistof aqueous emulsions in which the active substance is suspended.Suitable carriers for powders are for instance, rice starch and otherstarches; the bulk weight of the carriers may be made lighter, ifdesired, for example by adding highly dispersed silicic acid, or may bemade heavier by adding talcum. The tinctures may contain at least oneactive ingredient of formula I in aqueous ethanol, in particular 45% to75% ethanol, to which to of glycerol may be added, if desired. Solutionsprepared from polyethylene glycol and other conventional solubilitypromoters, and also optionally, from emulsifying agents, may be usedwith particular advantage in disinfecting healthy skin.

The content of active ingredient in pharmaceutical compositions forexternal application is preferably in the range of from 0.1% to 5%.

Gargles or concentrates for their preparation, and tablets for slowdissolution in the mouth, are suitable for the disinfection of the mouthand. throat. The former are preferably prepared from alcoholic solutionscontaining 1% to 5% of active substance to which glycerol or fiavouringsmay be added. Lozenges, that is solid dosage units, preferably have arelatively high content of sugar or similar substances and a relativelylow content of active substance, for instance 0.2% to 20% by weight, aswell as the usual conventional additives, such as binding agents andfiavourings.

Solid dosage units, in particular tablets, drages (sugar coated tablets)and capsules, are convenient for use in intestinal disinfection and forthe oral treatment of urinary tract infections. These units preferablycontain from 10% to 90% of the compound of formula I, to enable theadministration of daily dosages of from 0.1 to 2.5 grams to adults, orof suitably reduced doses to children. Tablets and drage cores areproduced by combining the compounds of formula I with solid, pulverulentcarriers such as lactose, saccharose, sorbitol, maize starch, potatostarch or amylopectin, cellulose derivatives or gelatines, preferablywith the addition of lubricants such as magnesium or calcium stearate orpolyethylene glycols of suitable molecular weight. Drage cores may thenbe coated, for example with concentrated sugar solutions which can alsocontain gum arabic, talcum and/or titanium dioxide, or they may becoated with a lacquer dissolved in volatile organic solvents or amixture of sol vents. Dyestuffs can be added to these coatings, forinstance to differentiate between varying dosages. Soft gelatinecapsules consist, for example, of a mixture of gelatines and glyceroland may contain, for example, mixtures of the compound of formula I withpolyethylene glycol. Hard gelatine capsules contain, for example,granulates of an active substance with solid pulverulent carriers, forinstance lactose, saccharose, sorbitol, mannitol, starches (such aspotato starch, maize starch or amylopectin), cellulose derivatives ofgelatines, and magnesium stearate or stearic acid.

In all forms of administration, compounds of the formula I can bepresent as sole active ingredients or they can also be combined withother known pharmacologically active, and especially anti-bacterialand/or anti-mycotically or other anti-microbially active substances, forexample to broaden the range of applications. They can be combined, forexample, with 5,7-dichloro- 2-methyl-8-quinolinol or other derivativesof S-quinolinol, with sulfamerazine or sulfafurazole or otherderivatives of sulfanilamide, with chloramphenicol or tetracycline orother antibiotics, with 3,4',5-tribromosalicylanilide or otherhalogenated salicylanilides, with halogenated carbanilides, withhalogenated benzoxazoles or benzoxazolones, withpolychloro-hydroxy-diphenylmethanes, with halogen-dihydroxy-diphenylsulphides, with 4,4'-dichloro- 2-hydroxy diphenylether or2,4,4'-trichloro-2-hydroxydiphenylether or otherpolyhalogenhydroxy-diphenylethers, or with bactericidal quaternarycompounds or with certain dithiocarbamic acid derivatives such astetramethylthiuram disulphide. Also, carriers which themselves havefavourable pharmacological properties may be used, for instance sulphur,as a powder base or zinc stearate as a component of ointment bases.

The invention also provides a method of protecting an organic materialsusceptible to bacterial or other microbial attack which comprisestreating the material with a compound of formula I. The organic materialmay be, for instance, a natural or synthetic polymeric material, aproteinaceous or carbohydrate substance, or a natural or synthetic fibreor textile material formed therefrom.

The invention also provides an animal feedtuff composition comprising acompound of formula I, in an amount sulficient to promote the growth ofthe animal fed with the composition.

The following examples will serve to further typify the nature of thepresent invention, but they, in no way, should be construed as alimitation on the scope thereof. The temperatures are given in degreesCentigrade.

EXAMPLE 1 (a) To a prepared mixture of 40 ml. of concentrated sulphuricacid and 40 ml. of ethanol are added 20 g. of 5-amino-4-cyano-1-methyl 3(5-nitro-2-furyl)-pyrazole and the resultant mixture is stirred on asteam bath at to C. for 90 minutes. The reaction mixture is then dilutedwith 500 ml. of water and stirring is continued until crystallisation iscomplete. The crude product was collected, washed with water andrecrystallised from water.

The product is S-amino 4 carbamoyl-1-methyl-3-(5-nitro-2-furyl)-pyrazole, having melting point 242 C. withdecomposition.

(b) The starting material 5-amino-4-cyano-1-methyl-3-(5-nitro-2-furyl)-pyrazole is prepared as follows:

To a stirred solution of 16.9 g. of 5-nitro-2-furaldehydeN'-methylhydrazone dissolved in 100 ml. of dimethylformamide is slowlyadded 17.8 g. of N-bromosuccinimide at 20-30 C. After further stirring,the mixture is cooled to 10 C.

To the stirred mixture is then added 6.6 g. of malononitrile followed bythe slow addition of a mixture of 10.1 g. of triethylamino and 25 ml. ofdimethylformamide at 1020 C.

After further stirring, the mixture is diluted with 500 ml. of icedwater and the precipitate is collected, Washed with water and dried. Thedried solid is recrystallised from ethyl acetate.

The product is 5-amino-4-cyano-1-methyl-3-(5-nitro- 2-furyl)-pyrazolehaving melting point 250 C. with decomposition.

In an analogous manner are prepared:

(c) 5-Amino-4-cyano-1-isopropyl-3-(5-nitro-2-furyl)- pyrazole;

(d) 5-Amino-4-cyano-1- (n-pentyl )-3-(5-nitro-2-furyl pyrazole;

(e) 5-Amino-4-cyano-1- 2-hydroxyethyl -3- (S-nitro- 2-furyl)-pyrazole,M.P. 216 C.;

(f) 5-Amino-4-cyano-1-ethyl-3-(5-nitro-2-furyl)- pyrazole, M.P. 189 C.;

(g) 5-Amino-4-cyano-1-n-propyl-3-(5-nitro-2-furyl)- pyrazole, M.P. C.

EXAMPLE 2 The procedure described in Example 1(a) is carried out using5-amino-4-cyano 1 isopropyl-3-(5-nitro-2- furyl)-pyrazole as startingmaterial instead of 5-amino4-cyano-1-methyl-3-(5-nitro-2-furyl)-pyrazole, the reaction conditionsbeing the same.

The product is 5-amino-4-carbamoyl-1-isopropyl-2-(5- nitro-2-furyl)-pyrazole.

7. EXAMPLE 3 The procedure described in Example 1(a) is carried outusing S-amino 4 cyano-1-(n-pentyl)-3-(5-nitro-2- furyl)-pyrazole asstarting material instead of S-amino-4-cyano-1-methyl-3-(5-nitro-2-furyl)pyrazole, the reaction conditionsbeing the same.

The product is 5-amino-4-carbamoyl-1-(n-pentyl)-3-(S-nitro-Z-furyl)-pyrazole.

EXAMPLE 4 The procedure described in Example 1(a) is carried out using 5amino 4-cyauo-l-(2-hydroxyethyl)-3-(5-nitro- 2 furyl)-pyrazole asstarting material instead of S-amino- 4 cyano1-methyl-3-(S-nitro-Z-furyl)-pyrazole, the reaction conditions being thesame.

The product is 5 amino 4-carbamoyl-1-(2-hydroxyethyl) 3(5-nitro-2-fury1)-pyrazole, having melting point 202 C.

EXAMPLE 5 (a) A mixture of 10 g. of 5 amino 4-cyano-1-(2- hydroxyethyl)3 (5 nitro 2 furyl) pyrazole and 200 ml. of acetic anhydride is heatedunder reflux for 4.5 hours and then evaporated to dryness under reducedpressure. The solid residue on recrystallisation from aqueousdimethylformamide gives S-acetamido-1-(2-acetoxyethyl)-4-cyano-3-(S-nitro-Z-furyl)-pyrazole, M.P. 191 C.

(b) A mixture of 12.0 g. of S-acetamido-1-(2-acetoxyethyl) 4cyano-3-(5-nitro-2-furyl)-pyrazole and 50 ml. of ethanolic hydrogenchloride (40% w./w.) is heated under refiux for 15 minutes and cooled.

To the mixture is then added 100 ml. of ice/water and the precipitatedsolid is collected, recrystallised from water and dried. The product is5-amino-4-carbamoyl-1- (2 hydroxyethyl) 3 (5-nitro-2-furyl)pyrazolehaving melting point 202 C.

EXAMPLE 6 To a solution of 23.3 g. of 5-amino-4-cyano-l-methyl-3-(5-nitro-2-furyl)-pyrazole dissolved in a mixture of 70 ml.dimethylformamide and 70 ml. pyridine is added 7.8 g. of acetyl chlorideand the mixture is heated under reflux for 2 hours. After cooling, themixture is diluted with 150 ml. of ice/water and 5acetamido-4-cyano-1rnethyl- 3-(5-nitro-2-furyl)-pyrazole which isprecipitated, collected, washed with water and dried, M.P. 250 C.

The procedure described in Example 5 is repeated using the 5 acetamido 4cyan-1-methyl-3-(5-nitro-2-furyl)- pyrazole as the starting materialinstead of S-acetamidol(2-acetoxyethyl)-4cyano-3-(5-nitro-2-furyl)-pyrazole, the reaction conditions being thesame.

The product is amino 4-carbamoyl-1-methyl-3-(5- nitro 2 furyl)-pyrazole,having melting point 245 C.

EXAMPLE 7 5 Acetamido-4-cyano-3-(5-nitro-2-furyl)-1-(n-propyl)- pyrazole(M.P. 215 C.) is prepared in a similar manner to 5 acetamido 4-cyanol-methyl-3-(5-nitro-2-furyl)- pyrazole described in Example 6, startingfrom S-amino- 4 cyano 3-(5-nitro-2-furyl)-1-(n-propyl)-pyrazole.

The procedure described in Example 5 is carried out using the 5acetamido 4-cyano-3-(5-nitro-2-furyl)-1- (n-propyl) -pyrazole asstarting material instead of 5- acetamido 1 (2 acetoxyethyl)4-cyano-3-(5-nitro-2- furyD-pyrazole, the reaction conditions being thesame.

The product is 5 amino 4-carbamoyl-3-(5-nitro-2- furyl) 1(n-propyl)-pyrazole, having melting point 198 C.

EXAMPLE 8 5 Acetamido 4 cyano-1-ethyl-3-(5-nitro-2-furyl)- pyrazole isprepared in a similar manner to 5 acetamido- 4cyano-l-methyl-3-(5-nitro-2-furyl)-pyrazole described in Example 6,starting from 5-amino-4cyano-1-ethyl-3- (5-nitro-2-furyl)-pyrazole.

Cir

The procedure described in Example 5 is carried out using 5 acetamido4-cyano-l-ethyl-3-(5-nitro-2-furyl)- pyrazole as starting materialinstead of S-acetamido-l- (2 acetoxyethyl)4-cyano-3-(5-nitro-2-fury1)-pyrazole, the reaction conditions being thesame.

The product is 5-amino-4-carbamoyl-1-ethyl-3-(5-nitro-2-furyl)-pyrazole, having melting point 210 C.

EXAMPLE 9 The procedure described in Example 1 is carried out using 5amino 4-cyanol-ethyl-3-(5-nitro-2-furyl)-pyrazole as starting materialinstead of 5-amino-4-cyano-lmethyl-3-(5-nitro-2-furyl)pyrazole, thereaction conditions being the same.

The product is 5 amino 4-carbamoyl-1-ethyl-3-(5-nitro-2-furyl)-pyrazole, having melting point 210 C.

EXAMPLE 10 The procedure described in Example 1 is carried out using 5amino-4-cyano-1-(n-propyl)-3-(5-nitro-2-furyl)- pyrazole as startingmaterial instead of 5-amino-4-cyano-1- methyl 3(5-nitro-2-furyl)-pyrazole, the reaction conditions being the same.

The product is 5-amino-4-carbamoyl-1-(n-propyl)-3-(5-nitro-2-furyl)-pyrazole, having melting point 198 C.

EXAMPLE 11 Preparation of Tablets A mixture consisting of g. of5-amino-4-carbamoyl- 1-methyl-3-(5-nitro-2-furyl)-pyrazole, 60.0 g. ofmaize starch and 35.0 g. of lactose is moistened with a solution of 5.0g. of gelatin and 3.0 g. of glycerol in 70.0 g. of water and granulatedthrough a sieve. The granulate is mixed with a mixture of 15.0 g. oftalcum, 10.0 g. of maize starch and 2.0 g. of magnesium stearate. Theresulting mixture is pressed into 1.000 tablets, each containing 100 mg.of active substance. If desired, the tablets can be grooved for betteradaption of the dosage.

EXAMPLE 12 Preparation of Drages Composition for 1.000 drages:

Composition I is granulated in the heat with composition II through asieve of 1.2 mm. mesh diameter. The dried granulate is mixed withcomposition III and the resulting mixture is pressed into 1.000 dragecores. These 9 are then coated with composition IV and dried. The dragesobtained weigh 255.0 mg. and contain 100 mg. of active substance.

EXAMPLE 13 Preparation of a Syrup The active substance and the colloidalsilicon dioxide are passed through a sieve of 1.2 mm. diameter (I).

The p-hydroxybenzoic acid esters, the citric and the sodium cyclamateare dissolved in the given amount of boiling distilled water; theglycerol is then added to this solution (II). The sodium carboxymethylcellulose and the sugar are thoroughly mixed (III).

Composition III is then added at 75 C. to solution H under stirringuntil complete dissolution of III. The viscous, slightly turbid liquidis cooled to room temperature, filtered, if necessary, and mixed withcomposition I. Water is added to the resulting mixture up to theprescribed weight of 1.155.0 g., and the syrup obtained is homogenized.

10 What is claimed is: 1. A pharmaceutical composition comprising anantibacterially effective amount of a compound of the formula I whereinR is alkyl having at most five carbon atoms or hydroxyalkyl having fromtwo to five carbon atoms, and a pharmaceutically acceptable carriertherefor.

2. The method for the treatment of a mammal suffering from a bacterialinfection selected from the group consisting of Staphylococcus,Escherichia coli, Klebsiella, and Salmonella, which method comprisesadministering to said mammal an antibacterially effective amount of thecomposition according to claim 1.

3. The method of protecting an organic material susceptible to bacterialattack, which comprises treating said material with an antibacteriallyeifective amount of a compound of the formula I SA ROSEN, PrimaryExaminer $2253? I UNITED STATES PATENT OFFICE v CERTIFICATE OFCORRECTION- Patent No. 3,830,926 natd August 20, 1974 hw g s GRAHAMARTON IHOWARTH ET AL ppears in the above-identified patent It iscertified that error a hereby corrected as shown below:

and that said Letters Patent are Column 1, line 9, ins e Great Britain,June 5,

rt Claims priority, applications 1969, 28423/69; April 28, 1970,.

Signed and sealed this 11th day of Februery 1975;

(SEAL) Attest:

C. MARSHALL DANN RUTH C. MASON Commissioner of Patents Attesting Officerand Trademarks aaa

